Bacterial infections remain among the most common and deadly causes of human disease. Infectious diseases are the third leading cause of death in the United States and the leading cause of death worldwide (Binder et al. (1999) Science 284, 1311-1313). Multi-drug-resistant bacteria now cause infections that pose a grave and growing threat to public health. It has been shown that bacterial pathogens can acquire resistance to first-line and even second-line antibiotics (Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998); Walsh, C. (2000) Nature 406, 775-781; Schluger, N. (2000) Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al., (2001) Ann. NY Acad. Sci. 953, 88-97). New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
RNA polymerase (RNAP) is the molecular machine responsible for transcription and is the target, directly or indirectly, of most regulation of gene expression (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Murakami, K. and Darst, S. (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov, S. and Nudler, E. (2003) Curr. Opin. Microbiol. 6, 93-100; Werner, F. (2007) Mol. Microbiol. 65, 1395-1404; Hirata, A. and Murakami, K. (2009) Curr. Opin. Structl. Biol. 19, 724-731; Jun, S., Reichlen, M., Tajiri, M. and Murakami, K. (2011) Crit. Rev. Biochem. Mol. Biol. 46, 27-40; Cramer, P. (2002) Curr. Opin. Struct. Biol. 12, 89-97; Cramer, P. (2004) Curr. Opin. Genet. Dev. 14, 218-226; Hahn, S. (2004) Nature Struct. Mol. Biol. 11, 394-403; Kornberg, R. (2007) Proc. Natl. Acad. Sci. USA 104, 12955-12961; Cramer, P., Armache, K., Baumli, S., Benkert, S., Brueckner, F., Buchen, C., Damsma, G., Dengl, S., Geiger, S., Jasiak, A., Jawhari, A., Jennebach, S., Kamenski, T., Kettenberger, Kuhn, C., Lehmann, E., Leike, K., Sydow, J. and Vannini, A. (2008) Annu. Rev. Biophys. 37, 337-352; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 671-685; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 686-704; Werner, F. and Grohmann, D. (2011) Nature Rev. Microbiol. 9, 85-98; Vannini, A. and Cramer, P. (2012) Mol. Cell 45, 439-446). Bacterial RNAP core enzyme has a molecular mass of ˜380,000 Da and consists of one β′ subunit, one β subunit, two α subunits, and one ω subunit; bacterial RNAP holoenzyme has a molecular mass of ˜450,000 Da and consists of bacterial RNAP core enzyme in complex with the transcription initiation factor σ (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami and Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov and Nudler (2003) Curr. Opin. Microbiol. 6, 93-100). Bacterial RNAP core subunit sequences are conserved across Gram-positive and Gram-negative bacterial species (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 671-685; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 686-704). Eukaryotic RNAP I, RNAP II, and RNAP III contain counterparts of all bacterial RNAP core subunits, but eukaryotic-subunit sequences and bacterial-subunit sequences exhibit only limited conservation (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Cramer, P. (2004) Curr. Opin. Genet. Dev. 14, 218-226; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 671-685; Lane, W. and Darst, S. (2010) J. Mol. Biol. 395, 686-704).
Crystal structures have been determined for bacterial RNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824; Cramer et al., (2000) Science 288, 640-649; Cramer et al., (2001) Science 292, 1863-1876).
Structures also have been determined for RNAP complexes with nucleic acids, nucleotides and inhibitors (Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363; Campbell, et al. (2005) EMBOJ. 24, 674-682; Tuske, et al. (2005) Cell 122, 541-522; Temiaov, et al. (2005) Mol. Cell 19, 655-666; Mukhopadhyay, J., Das, K., Ismail, S., Koppstein, D., Jang, M., Hudson, B., Sarafianos, S., Tuske, S., Patel, J., Jansen, R., Irschik, H., Arnold, E., and Ebright, R. (2008) Cell 135, 295-307; Belogurov, G., Vassylyeva, M., Sevostyanova, A., Appleman, J., Xiang, A., Lira, R., Webber, S., Klyuyev, S., Nudler, E., Artsimovitch, I., and Vassylyev, D. (2009) Nature. 45, 332-335; Vassylyev, D., Vassylyeva, M., Perederina, A., Tahirov, T. and Artsimovitch, I. (2007) Nature 448, 157-162; Vassylyev, D., Vassylyeva, M., Zhang, J., Palangat, M., Artsimovitch, I. and Landick, R. (2007) Nature 448, 163-168; Gnatt, et al. (2001) Science 292, 1876-1882; Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al. (2004b) Cell 119, 481-489; Ketenberger, et al. (2004) Mol. Cell 16, 955-965; Bushnell, et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 1218-1222; Kettenberger, et al. (2005) Natl. Structl. Mol. Biol. 13, 44-48; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723).
Bacterial RNAP is a proven target for antibacterial therapy (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007) Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide, L., Gualtieri, M. and Leonetti, J. (2007) Drug Discov. Today 12, 200-208; Mariani, R. and Maffioli, S. (2009) Curr. Med. Chem. 16, 430-454; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723; Srivastava, A., Talaue, M., Liu, S., Degen, D., Ebright, R. Y., Sineva, E., Chakraborty, A., Druzhinin, S., Chatterjee, S., Mukhopadhyay, J., Ebright, Y., Zozula, A., Shen, J., Sengupta, S., Niedfeldt, R., Xin, C., Kaneko, T., Irschik, H., Jansen, R., Donadio, S., Connell, N. and Ebright, R. H. (2011) Curr. Opin. Microbiol. 14, 532-543). The suitability of bacterial RNAP as a target for antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP subunit sequences are conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP subunit sequences are only weakly conserved in eukaryotic RNAP I, RNAP II, and RNAP III (providing a basis for therapeutic selectivity).
The rifamycin antibacterial agents—notably rifampin, rifapentine, and rifabutin—function by binding to and inhibiting bacterial RNAP (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723; Floss and Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005) Cell 122, 351-363; Feklistov, A., Mekler, V., Jiang, Q., Westblade, L., Irschik, H., Jansen, R., Mustaev, A., Darst, S., and Ebright, R. (2008) Proc. Natl. Acad. Sci. USA 105, 14820-14825). The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and prevent the extension of RNA chains beyond a length of 2-3 nt.
The rifamycins are in current clinical use in treatment of Gram-positive and Gram-negative bacterial infections (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723; Floss and Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912). The rifamycins are first-line treatments for tuberculosis and are the only current first-line treatments for tuberculosis able to kill non-replicating tuberculosis bacteria, to clear infection, and to prevent relapse (Mitchison, D. (2000) Int. J Tuberc. Lung Dis. 4, 796-806). The rifamycins also are first-line treatments for biofilm-associated infections of catheters and implanted medical devices and are among the very few current antibacterial drugs able to kill non-replicating biofilm-associated bacteria (Obst, G., Gagnon, R. F., Prentis, J. and Richards, G. K. (1988) ASAIO Trans. 34, 782-784; Obst, G., Gagnon, R. F., Harris, A., Prentis, J. and Richards, G. K. (1989) Am. J. Nephrol. 9, 414-420; Villain-Guillot, P., Gualtieri, M., Bastide, L. and Leonetti, J. P. (2007) Antimicrob. Agents Chemother. 51, 3117-3121.
The clinical utility of the rifamycin antibacterial agents is threatened by the emergence and spread of bacterial strains resistant to known rifamycins (Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723; Floss and Yu (2005) Chem. Rev. 105, 621-632; Campbell, et al. (2001) Cell 104, 901-912). Resistance to rifamycins typically involves substitution of residues in or immediately adjacent to the rifamycin binding site on bacterial RNAP—i.e., substitutions that directly decrease binding of rifamycins. A significant and increasing percentage of cases of tuberculosis are resistant to rifampicin (1.4% of new cases, 8.7% of previously treated cases, and 100% of cases designated multidrug-resistant, in 1999-2002; Schluger, N. (2000) Int. J. Tuberc. Lung Dis. 4, S71-S75; Raviglione, et al. (2001) Ann. N Y. Acad. Sci. 953, 88-97; Zumia, et al. (2001) Lancet Infect. Dis. 1, 199-202; Dye, et al. (2002) J. Infect. Dis. 185, 1197-1202; WHO/IUATLD (2003) Anti-tuberculosis drug resistance in the world: third global report (WHO, Geneva)). Strains of bacterial bioweapons agents resistant to rifampicin can be, and have been, constructed (Lebedeva, et al. (1991) Antibiot. Khimioter. 36, 19-22; Pomerantsev, et al. (1993) Antibiot. Khimioter. 38, 34-38; Volger, et al. (2002) Antimicrob. Agents Chemother. 46, 511-513; Marianelli, et al. (204) J. Clin. Microbiol. 42, 5439-5443).
In view of the public-health threat posed by rifamycin-resistant bacterial infections, there is an urgent need for new antibacterial agents that target bacterial RNAP and an especially urgent need for new antibacterial agents that target bacterial RNAP derivatives resistant to known rifamycins. (See Darst, S. (2004) Trends Biochem. Sci. 29, 159-162; Chopra, I. (2007) Curr. Opin. Investig. Drugs 8, 600-607; Villain-Guillot, P., Bastide, L., Gualtieri, M. and Leonetti, J. (2007) Drug Discov. Today 12, 200-208; Mariani, R. and Maffioli, S. (2009) Curr. Med. Chem. 16, 430-454; Ho, M., Hudson, B., Das, K., Arnold, E. and Ebright, R. (2009) Curr. Opin. Structl. Biol. 19, 715-723; Srivastava, A., Talaue, M., Liu, S., Degen, D., Ebright, R. Y., Sineva, E., Chakraborty, A., Druzhinin, S., Chatterjee, S., Mukhopadhyay, J., Ebright, Y., Zozula, A., Shen, J., Sengupta, S., Niedfeldt, R., Xin, C., Kaneko, T., Irschik, H., Jansen, R., Donadio, S., Connell, N. and Ebright, R. H. (2011) Curr. Opin. Microbiol. 14, 532-543.)